Electron paramagnetic resonance (EPR) has been used to make direct measurements of reaction kinetics, particularly for those reactions involving free radicals. Exemplary applications of stopped-flow EPR include studies of: the kinetics of enzymatic molecular oxygen consumption; kinetics of micromolar quantities of spin-trapped free radicals followed on a sub-second time scale; time resolved protein folding and unfolding; and oxygenation of various organic molecules as performed in research concerning the effects of free radicals in ischemia.
Many EPR spectrometers utilize a conventional cavity resonator similar to one described in Yamazaki et al., J. Biol. Chem., 235, 2444 (1960). These resonators are characterized generally by a rectangular metallic structure or frame, the inside of which is a resonant cavity through which a capillary or flat cell may extend. These conventional cavity resonators are typically ill suited for the study of lossy dielectric samples, which includes most biologicals and solutions of free radicals. The sample volumes utilized by the conventional cavity resonators are measured by the milliliter. However, biological samples are often limited in supply which presents a particularly troublesome problem since employing a conventional cavity resonator to study transient processes usually requires large volumes of relatively concentrated material. Another problem arises from the failure of the conventional cavity resonator to effectively isolate the region of microwave electric field (E.sub.1) from the region of microwave magnetic fields (H.sub.1), the latter of which induces the desired EPR transitions. The inability to separate the E.sub.1 and H.sub.1 components is an important characteristic since the electric field may often interact with a sample to cause resonant frequency changes and Q losses (Q is the quality factor, either calculated as being 2.pi..times.microwave energy stored by the device/energy dissipated per cycle of microwaves or calculated as the resonant frequency (.nu..sub.o) of the device/the difference in frequency (.DELTA..nu.) obtained at the 3 dB half power absorbing points on the mode pattern of the device). This undesirable interaction between the sample and the E.sub.1 component is especially pronounced with lossy dielectric samples.
A design more recently used today for continuous and stopped flow EPR is based on a loop gap resonator (LGR) as described in Hubbell et al. Rev. Sci. Instrumen., 58, 1879 (1987). The standard design for an LGR utilizes a machined MACOR.RTM. ceramic block having two holes extending through the block, these holes are connected by a thin slit extending through said block, the interior of the holes and slit are plated with silver. Unlike the conventional cavity resonators the LGR utilizes a much smaller sample volume, however, due to the complex configuration of the LGR and its small components the LGR based EPR probe is typically susceptible to a significant loss of sensitivity with use. In addition, due to the configuration of the loop and gap areas of the LGR low Q is experienced due to electric field (E.sub.1) interaction with lossy dielectric samples. In addition, due to the design of the LGR, flow and stopped-flow induced noise is a limiting factor when utilizing stopped flow technology since repetitive starting and stopping of the sample flow in the capillary is required. This forced movement within the capillary tube creates vibrations which effectively limit the sensitivity of the LGR. In addition, the structure of the LGR makes it difficult to assemble and disassemble the device. In the event any particular part becomes contaminated or worn, the ability to replace or repair any individual component takes considerable effort and often requires returning the part to the manufacturer. In addition, the use of delicate and complex machined parts results not only in less durable parts but in expensive replacement parts. Furthermore, variable capacitance coupling used in connection with the LGR probe often causes large resonance frequency changes when the coupling is changed. The resulting simultaneous coupling and frequency changes greatly complicate attaining critical coupling.
Many EPR spectroscopy systems, such as existing Bruker systems, which lack the GaAsFET amplifier often have significant difficulty in making their AFC (automatic frequency control) lock to a low Q resonator. This difficulty is more commonly experienced at low powers. Difficulty in obtaining an AFC lock may cause frequency drift, drift in AFC error voltage, uncertain admixture of absorption and dispersion and noise. A higher Q system makes it easier to obtain an AFC lock without GaAsFET amplifications.
Therefore there exists a need for an EPR probe having a high Q, in particular, one suitable for use in the study of highly lossy samples and that is also capable of maintaining a high Q value when studying such samples. In addition, there exists a need for an EPR probe which utilizes small sample volumes but is capable of withstanding the vibrations created by stopped flow techniques and capable of maintaining a high Q and its sensitivity. In addition, there exists a need for an EPR probe having a simple and durable design which is readily assembled and disassembled and capable of quick and inexpensive repair.